Methods for determining protein binding specificity using peptide libraries

ABSTRACT

A method for determining protein binding specificity using a screen of a peptide library is provided. The method can be used to determine binding specificity for human NAD + -dependent deacetylase SIRT1, and to identify the most efficiently deacetylated peptide sequences. The method can be also used to screen a combinatorial H4 histone N-terminal tail peptide library to examine the binding preferences of a α-phos (S1) H4 antibody toward all known possible H4 histone modification states.

CROSS-REFERENCE TO RELATED APPLICATIONS

This invention claims priority to U.S. Provisional Patent ApplicationSer. No. 60/729,866, filed Oct. 24, 2005.

GOVERNMENT INTERESTS

This invention was made with United States government support awarded bythe NIH, grants Nos. GM065386 and GM059785. The United States may havecertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a method for determining substrate specificityof protein binding using peptide libraries.

BACKGROUND OF THE INVENTION

An emerging paradigm for discovery in pharmaceutical and relatedbiotechnology is the assembly of novel synthetic compound libraries bynew methods of solid phase “combinatorial” synthesis. Combinatorialchemistry refers to a set of strategies for the parallel synthesis ofmultiple compounds or compounds mixtures, either in solution or on solidsupports in the form of polymer-based resins (“beads”).

One implementation of combinatorial synthesis that is suitable toproduce large chemical libraries relies on “one-bead-one-compound”(OBOC) libraries, which contain from 10⁶ to 10⁸ compounds. Theselibraries are screened by performing a variety of chemical andbiochemical assays to identify individual compounds eliciting aresponse. The chemical identity of such specific compounds can then bedetermined by direct analysis, using, e.g., micro-sequencing and massspectrometry.

In peptidic OBOC libraries, peptide sequences from a group of selectedamino-acid building blocks are represented in an on-bead format in whichmany copies of only one sequence exist on each bead. OBOC librariespermit sifting through a list of peptide substrate sequences tocorrelate top hits with protein sequence databases. This strategy hasbeen used successfully, for example to determine the optimal peptidesubstrates of peptide deformylase, a Fe²⁺ metalloenzyme that catalyzesN-terminal deformylation of nascent polypeptides in eubacteria (Hu etal., 1999, Biochemistry 38: 643-650).

Histone proteins serve to package DNA and to regulate its accessibilityfor processes including transcription, repair and replication. Six majorhistone classes are known. Two each of the class H2A, H2B, H3 and H4,assemble to form one nucleosome core particle around which DNA iswrapped. Acting as spools around which DNA winds, histones play a rolein gene regulation. Histones achieve this control over DNA by acting assubstrates to a host of posttranslational modifications that dictatefunction. Particularly dense with posttranslational information are theN-terminal histone tails. These can be covalently modified at severalsites. Modifications of the histone tail include methylation,acetylation, phosphorylation, ubiquitination, sumoylation,citrullination, and ADP ribosylation. The core of the histones can alsobe modified. Combinations of modifications are thought to constitute acode, the so-called “histone code”. The histone code hypothesis assertsthat histone-binding proteins and histone-modifying enzymes read andinterpret the posttranslational states of properly “primed” histones tofacilitate a particular outcome such as gene silencing, transcription,mitosis, etc. (Strahl et al., 2000, Nature 403: 41-45). However, thusfar, the combinatorial complexity of the histone modification patternshas precluded a systematic inquiry of the patterns recognized by these“code readers”, proteins and enzymes that would display preferentialspecificity for these context-dependent modifications.

Protein deacetylases have been implicated in a variety of disease statesincluding aging, diabetes, HIV regulation, cancer, cardiovasculardisorders, and neurodegenerative diseases. Histone deacetylaseinhibitors are currently in clinical trials as cancer treatments. Inparticular, the Silent information regulator 2 (Sir2) family of NAD⁺dependent protein deacetylases has been studied in recent years. Thisburgeoning interest can be attributed to the important roles of Sir2enzymes (sirtuins) in regulating chromatin architecture, promotingtranscriptional silencing and longevity, and in fatty acid metabolism.NAD⁺ dependent lysyl deacetylation is characterized by thestoichiometric release of nicotinamide and a novel metabolite,O-acetyl-ADP-ribose (OAADPr).

The Sir2 family of deacetylases is highly conversed among all forms oflife with seven known human homologs (SIRT1-7). The most studiedmammalian homolog, SIRT1, is a nuclear enzyme that has been found todeacetylate a number of proteins. Histones H3 and H4, p53, p300, TAF₁68,PCAF/MyoD, PGC-1 alpha, FOXO1 and 4, NF-kappaB, and Tat are examplesreported to be either biological targets and/or in vitro substrates ofSIRT1. Collectively, the variety of proposed physiological targetsreflects the functional diversity of SIRT1.

Identifying biological substrates is an important step in understandingthe molecular basis for sirtuin phenotypes. However, in many of thestudied cases, a certain degree of logical bias was used to link thetarget protein and SIRT1, as unbiased global substrate screeningprocedures were not used. Varying conclusions have been reached inregard to sirtuin substrate specificity and recognition. Most strikingare the conclusions that sirtuins display minimal side-chain recognition(Avalos et al., 2002, Mol. Cell. 10: 523-535; Zhao et al., 2003,Structure (Camb) 11: 1403-1411) and that SIRT1 displays no substratesequence specificity (Blander et al., 2005, J. Biol. Chem. 280:9780-9785). In contrasting reports, clear substrate preferences werenoted for yeast Sir2 and HST2 (Borra et al., 2004, Biochemistry 43:9877-9887), and human SIRT2 (North et al., 2003, Mol. Cell. 11:437-444).

To date, only one study has attempted to probe sirtuin substratespecificity using an acetyl-peptide library approach (Blander et al.,2005). Curiously, the study reported that SIRT1 displayed no substratespecificity in vitro, a conclusion based on an oriented peptide library.With this method, only globally preferred amino-acids could be resolved,and the actual sequence of individual peptides was not elucidated.Although the peptide library technique has been successful for examiningprotein kinase substrate specificity (Songyang et al., 1994, Curr. Biol.4: 973-982), its usefulness toward protein deacetylases remainsuncertain.

SUMMARY OF THE INVENTION

This invention provides a method for determining binding specificity ofa protein deacetylase, which includes contacting the protein deacetylasewith a peptide library. The peptide library includes a plurality ofsolid phase supports, where each solid phase support is linked to adifferent and distinct peptide. The method includes labeling the peptidelibrary with a label specific for an amino group formed upondeacetylation, and correlating the label intensity with bindingspecificity of the protein deacetylase.

The label can be specific for an amino group formed upon deacetylation.The solid phase supports used to practice the method can be beads. Thelabel can be colorimetric, radioactive, or fluorescent. The label canalso be a labeled quantum dot.

The method can include sorting the solid phase supports from the labeledpeptide library on the basis of label intensity. The method can includedetermining the sequence of the peptide attached to the solid phasesupport.

The protein deacetylase assayed by the method can be a sirtuin.Preferably, the protein deacetylase is SIRT1.

The peptide sequence can be selected from the group consisting of:LNKDQ, WHKFQ, WHKFE, SYKQW, QPKQI, VQKII, HRKMP, HKKMP, AVKFM, NHKLL,RFKPE, KFKPE, FEKYR, MMKQQ, WGKSP FEKYK, WPKWQ, RAKMD, KAKMD, GTKTG,GYKPT, IFKTF, TEKQE, HWKTH, DSKGA, SDKYH, NHKII, WWKHG, PIKEQ, RPKQF,KPKQF, DVKMH, IYKND, TPKNA, PGKLY, RWKIT, KWKIT, WRKIT, WKKIT,WPKITPWKIT, RPKSI, KPKSI, PRKSI, and PKKSI.

This invention provides a method for determining binding specificity ofan enzyme. The method includes contacting a combinatorial peptidelibrary with the enzyme, where the peptide library comprises a pluralityof solid phase supports, where each solid phase support is linked to adifferent and distinct peptide, labeling peptides with a label specificfor peptides covalently modified by the enzyme, and correlating theintensity of the label with peptides that are covalently modified by theenzyme, thereby determining binding specificity of the enzyme. Thecovalent modification may include a post-translational modification.

This invention provides an analytical method, which includes generatinga combinatorial library of peptides that includes one or more peptidesequences attached to a solid phase support, where the combinatoriallibrary includes a plurality of solid phase supports linked to adifferent and distinct peptide, and where each peptide comprises two ormore chemically modified amino acids, contacting the combinatoriallibrary with a protein, detecting the protein bound to one or morepeptides using a label, and correlating the label intensity withpeptides to which the protein binds, thereby determining the bindingspecificity of the protein. The solid phase supports can be beads. Thelabel can be calorimetric, radioactive, or fluorescent. The label can bea labeled quantum dot. The solid phase supports from the labeled peptidelibrary can be sorted on the basis of label intensity.

The method can include determining the sequence of the peptide attachedto the solid phase support. The method can include determining themodification status of the peptide attached to the solid phase support.

Each peptide sequence can include at least 5 amino acids. At least onepeptide sequence can include covalently modified amino acid.

The covalent modification can include methylation, acetylation,phosphorylation, ubiquitination, sumoylation, citrullination, or ADPribosylation. The protein that is used to contact the combinatorialpeptide library can be an enzyme. The protein that is used to contactthe combinatorial peptide library can be an antibody. The combinatoriallibrary can include one or more N-terminal peptide sequences from ahistone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing data from differential biotinylationexperiments with quantum dot tagging.

FIG. 2 is an image of a fluorescence micrograph showing data fromdifferential biotinylation experiments with quantum dot tagging.

FIG. 3 depicts parts of the capping scheme used for the identificationof randomized peptide sequences: (1, top) chemical structure of thecapping reagent phenylacetic acid; (2, bottom) chemical structure of thecapping reagent pentenoic acid.

FIG. 4 illustrates the capping strategy used during peptide synthesisfor generation of a peptidic “mass ladder”.

FIG. 5 schematically depicts a quantum dot bead-based assay.

FIG. 6 is a graph showing the fluorescence distribution of librarymembers.

FIG. 7 is a graph showing the mass spectrum obtained for microsequencingof a top hit peptide sequence.

FIG. 8 schematically depicts the on-bead Western analysis. Beads withphosphorylated sequences (top) or unphosphorylated sequences (bottom)corresponding to the N-terminal tails of histone H4 were assayed.

FIG. 9 shows images of fluorescence micrographs of the on-bead Westernanalysis. Left panel, fluorescent microscopic image ofAcSGRGKGG(AcK)GLG(AcK)GGAKRHRKVBBM-Macrobead (1) (SEQ ID NO:1) after theon-bead assay. Center panel, fluorescent microscopic image ofAcpSGRGKGG(AcK)GLG(AcK)GGAKRHRKVBBM-Macrobead (2) (SEQ ID NO:2). Rightpanel, fluorescent microscopic image of a 5:1 ratio of (1) to (2). Brefers to beta-alanine.

FIG. 10 illustrates the H4 histone N-terminal tail library, which iscomprised of the sequence (SEQ ID NO:71) corresponding to the firsttwenty-one amino acids of human histone H4 attached to a linker composedof two β-alanines (B) and a methionine (M).

FIG. 11 is an image of a fluorescence micrograph showing the results ofa H4 histone N-terminal tail library screen with a α-phos (S1) H4antibody.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of peptide synthesis, molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, immunology, protein kinetics, and mass spectroscopy, whichare within the skill of art. Such techniques are explained fully in theliterature, e.g. in Bodanszky et al., 1976, Peptide Synthesis, 2^(nd)ed., John Wiley and Sons; Sambrook et al., 2000, Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press; CurrentProtocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc.;Kriegler, 1990, Gene Transfer and Expression: A Laboratory Manual.Stockton Press, New York.; Dieffenbach et al., 1995, PCR Primer: ALaboratory Manual, Cold Spring Harbor Laboratory Press, each of which isincorporated herein by reference in its entirety.

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally, enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. Procedures employing commercially available assay kitsand reagents are typically used according to manufacturer-definedprotocols unless otherwise noted.

The terms “a”, “an”, “the” and the like, unless otherwise indicated,include plural forms.

The term “acetyl”, sometimes called “ethanoyl”, is a functional group,the acyl of acetic acid, with chemical formula —COCH₃.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, colorimetric labels,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or quantum dots. As used herein, the term “label”also includes indirect labeling of proteins using detectable labelsbound to other molecules or complexes of molecules that bind to aprotein of interest, including antibodies and proteins to which antiseraor monoclonal antibodies specifically bind. As used herein, the term“colorimetric label” includes a label that is detected using anenzyme-linked assay.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, and single chain antibodies, as well as Fabfragments, including the products of a Fab or other immunoglobulinexpression library. With respect to antibodies, the term,“immunologically specific” refers to antibodies that bind to one or moreepitopes of a protein of interest, but which do not substantiallyrecognize and bind other molecules in a sample containing a mixedpopulation of antigenic biological molecules.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A peptide or protein that is thepredominant species present in a preparation is substantially purified.The term “purified” denotes that a peptide or protein gives rise toessentially one band in an electrophoretic gel or HPLC spectrum.Particularly, it means that the peptide or protein is at least 85% pure,more preferably at least 95% pure, and most preferably at least 99%pure.

Two peptides or polypeptides are said to be “identical” if the sequenceof amino acid residues in the two sequences is the same when aligned formaximum correspondence as described below.

Peptide or protein sequence identities are evaluated using the BasicLocal Alignment Search Tool (“BLAST”) which is well known in the art(Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268;Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLASTprograms can be used with the default parameters or with modifiedparameters provided by the user.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the peptide or polypeptide sequence in the comparison windowmay comprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of amino acid sequences for purposes ofthis invention normally means peptide or polypeptide sequence identityof at least 40%. Preferred percent identity of peptides or polypeptidescan be any integer from 40% to 100%. More preferred embodiments includeat least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.

Peptides or polypeptides that are “substantially similar” sharesequences as noted above except that residue positions which are notidentical may differ by conservative amino acid changes. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, asparticacid-glutamic acid, and asparagine-glutamine.

The term “combinatorial library” refers to a collection of compoundssynthesized in parallel or as a collection of compounds synthesized withmixtures of reagents or employing a split-and-mix methodology from a setof defined building blocks and using these building blocks in manycombinations to generate a complex library of novel compounds. Oneimplementation of combinatorial synthesis is the generation of“one-bead-one-compound” (OBOC) libraries, where each novel compound isrepresented on a single bead. These libraries can be screened byperforming a variety of chemical and biochemical assays to identifyindividual compounds eliciting a response. The identity of the compoundon the support is either known by prior knowledge from direct parallelsynthetic procedures, or is determined by direct analysis afterdetection using, e.g., micro-sequencing and mass spectrometry.

In peptidic OBOC libraries, peptide from a group of selected amino-acidbuilding blocks can be represented in an on-bead format in which manycopies of only one sequence and of only one post-translationallymodified state exist on each bead. Alternatively, peptides may beattached to other suitable types of support, including microarrays,microplates, chips, or other surfaces that are suitable for detectingprotein binding events.

The invention described here uses both modified and unmodified aminoacids as building blocks to create combinatorial peptide libraries toevaluate the binding specificities of proteins. These peptide librariescan vary in amino acid sequence and in modification state, e.g.methylation, acetylation or phosphorylation. The chemical identity(amino-acid sequence and post-translational status) of these peptidescan be determined by direct analysis, using, e.g., micro-sequencing andmass spectrometry. Identification of the peptide sequences and theirpost-translational status can be performed before a protein bindingassay is conducted. Alternatively, identification of the peptidesequences and their post-translational status can be performed before aprotein binding assay is conducted. As well, identification of thepeptide sequences and their post-translational status can be performedboth before and after a protein binding assay is conducted.

The method includes screening a peptide library. In one example, themethod provides for the use of a “one-bead-one-compound” (OBOC) peptidelibrary, also known as OBOC combinatorial library. Examples of suchlibraries are described in Lam et al., 1991, Nature 354: 82-84, and inFurka et al., 1991, Int. J. Pept. Protein Res. 37: 487-493.

The OBOC combinatorial library method synthesizes 10²-10⁸ of randomcompounds such that each bead displays only one compound. Bead librariesare screened, and positive beads are isolated for structure analysis.Peptide substrates and inhibitors of protein kinases, and peptideligands for cell surface receptors can be identified using this method(Lam et al., 2003, Acc. Chem. Res. 36: 370-377).

A peptide library can be synthesized on various types of solid supportsusing methods known in the art, for example those described in U.S. Pat.Nos. 7,122,323 and 5,510,240. Preferably, the library is synthesizedusing beads as solid phase support. In one example, the solid supportcan have the form of beaded resin (“beads”).

The peptide library can be an OBOC acetyl-peptide library. One or moreof the amino acids from the peptide can be deprotected using methodsknown in the art, e.g. trifluoroacetic acid. One or more of the aminoacids from the peptide can be acetylated. In addition to acetylation ofat least one amino acid, one or more of the amino acids can be modifiedin a variety of ways, for example through covalent modifications.Modifications can include post-translational modifications orintroduction of non-classical amino acids, as described in U.S. Pat. No.5,510,240.

The length of the peptide chain can vary. Preferably, the OBOCacetyl-peptide library is generated using 5-mer peptides, i.e. peptideswith 5 amino acid residues. One or more of these 5 amino acids from thepeptide can be acetylated. Preferably, the central (third) amino acid isacetylated. More preferably, the central, acetylated amino acid islysine. Preferably, unique sequences are constructed around a centralepsilon-amino acetylated lysine.

When the peptide library is an acetyl-peptide library, the method can beused for determining protein deacetylase substrate specificity. Theprotein deacetylase can belong to the sirtuin family. Preferably, theprotein deacetylase is SIRT1.

The peptide library can be a combinatorial peptide library based onN-terminal histone sequences. In this example, one or more of thepeptide sequences include N-terminal histone sequences. Preferably, thecombinatorial OBOC peptide library is based on N-terminal amino acidsequence of histone H4. The N-terminal histone sequences can have adifferent number of amino acid residues. Preferably, the N-terminalhistone sequences are 21-mers, i.e. they have 21 amino acid residues.These sequences can be attached to solid support directly, or via one ormore amino acids that act as linkers.

The N-terminal histone sequences can be modified to include variouspost-translational modifications. The modifications can be covalent. Themodifications can include, for example, methylation, acetylation,phosphorylation, ubiquitination, sumoylation, citrullination, or ADPribosylation. Modifications can also include the introduction ofnon-classical amino acids. Each N-terminal histone sequence can includeone or more of these modifications.

When the combinatorial peptide library is based on N-terminal histonesequences, the invention provides methods for determining and evaluatinghistone-binding proteins or histone-modifying enzymes. Preferably, thehistone-binding protein is an antibody. More preferably, thehistone-binding protein is an α-phos (S1) H4 antibody.

Prior to library construction, peptide length may initially beconsidered to determine whether relatively short peptides would functionas efficient protein substrates. For example, prior to libraryconstruction, peptide length may be considered to determine whetherrelatively short acetyl-peptides would function as efficient enzymaticsubstrates. Also, for example, prior to library construction, peptidelength may be considered to determine the preferred length of theN-terminal amino acid chain of a histone and its efficiency as a proteinsubstrate.

The method provides for the use of a quantum dot tagging, i.e. labelingstrategy. Quantum dots are nanoparticles that exhibit exquisitephotochemical properties owing to their semiconductor cores and areemerging as ideal fluorophores for screening OBOC libraries (Falciani etal., 2005, Chem. Biol. 12: 417-426). These properties include robustphotostability, high quantum yield, and a sharp emission with a broadrange of excitation wavelengths. Coupled with a bead-sorting instrument,quantum dots allow the screening of hundreds of thousands of peptidesequences for protein binding and protein activity in a single day.Quantum dots can be used, for example, to label the protein that is incontact with a peptide that is attached to a bead.

The method provides for sorting of beads. Bead sorting can be performedmanually or it can be automated. Bead sorting is preferably performedbased on beads that are labeled. Bead labeling can be performed using avariety of methods known in the art. Preferably, beads can be labeledwith a fluorescent label or with any other type of label. When beads arelabeled with a fluorescent label, then the method can includefluorescent bead-sorting. In one example, a protein that is assayed andthat specifically binds to beads, can be labeled. In another example,beads with peptides deacetylated by a protein deacetylase can belabeled. For example, these beads can be first biotinylated and thentagged (labeled) with streptavidin-coated quantum dots.

After bead-sorting, peptides sequences and their correspondingpost-translationally-modified status can be extracted from individualbeads in the library. The sequences and modification state of thesepeptides can then be identified. Identification of the peptides can beperformed, for example, by mass spectrometry, or by micro-sequencing.Identification of the peptides can thus identify the particularsequences and modification state for which the assayed protein showsconsiderable binding preference.

A peptide library that is already spatially-addressed, e.g. eachsequence and modification state is known at each support (e.g. bead),can be screened by the methods described.

The present invention provides a high-throughput method for determiningsubstrate specificity of protein deacetylases using a one-bead,one-compound OBOC acetyl-peptide library with a quantum dot taggingstrategy and automated bead-sorting. The OBOC acetyl-peptide librarymethod allows context-specific identification of preferred peptidesubstrates. This is in contrast to the previously published approachthat can only uncover globally-preferred amino-acids at each position.The OBOC acetyl-peptide library method can be applied to anyhistone/protein deacetylase from class I, II or III.

Various applications of peptide libraries of this type can beenvisioned. For example, the sequence information obtained from thislibrary can be used to generate acetyl-peptide specific antibodies forWestern blot analysis. This can provide in vivo validation ofacetylation at protein acetylation sites discovered in BLAST searchesand sequence comparisons. Such antibodies could also be employed inimmunoprecipitation studies. Mass spectral analysis could then beperformed to identify the acetylated proteins. Identification ofenzymatic substrates, i.e. the sequence information obtained from thelibrary, can also be used to generate acetyl-peptide specificinhibitors.

Other uses of the library include the creation of super-substrates forthe in vivo generation of O-acetyl-ADP-ribose (OAADPr) to elucidate itscellular roles. Co-crystal studies could be executed to uncover how Sir2interacts with these optimal substrate sequences. Limited peptidesubstrates co-crystallized with Sir2 have shown interactions primarilywith the peptide backbone (Avalos et al., 2002, Mol. Cell. 10: 523-535).However, the method of this invention provides for the use of side-chaininteractions to bind and catalyze protein deacetylation.

Hits from libraries of this type could serve as starting points for thedesign of peptidomimetics for a variety of applications, e.g. for use aspotential therapeutics (Nefzi et al., 2004, J. Org. Chem. 69: 3603-3609;Falciani et al., 2005, Chem. Biol. 12: 417-426). Optimized substratesreflect higher binding affinity to a protein, e.g. enzyme. Modificationof the peptide to prohibit enzymatic turnover and protease degradationcould be implemented to generate a specific, tight-binding in vivoinhibitor.

The original peptide sequences can be further modified to confer alteredchemical and biological properties (Nefzi et al., 2004, J. Org. Chem.69: 3603-3609; Falciani et al., 2005, Chem. Biol. 12: 417-426). Thisstrategy has been used to tailor-make peptides into therapeutics thatavoid the pitfalls of proteolytic cleavage, rapid clearance from thecirculatory system, inability to pass through the blood brain barrier,and lack of oral activity (Nefzi et al., 2004).

This invention provides a method for identification of the molecularrecognition events involved in the histone code via OBOC combinatorialpeptide libraries based on N-terminal histone sequences. This methodprovides for synthesis and evaluation of all possible permutations (atknown modification sites) of the 21 N-terminal amino acids of histoneH4. In a preferred embodiment, the evaluation of the binding specificityis performed using an antibody directed to serine phosphorylation and aquantum dot detection strategy (Garske and Denu, 2006, Biochemistry 45:94-101; Kodadek et al., 2006, Mol. Biosyst. 2: 25-35). However, thegeneral approach is applicable to interrogating the preferences of anyhistone-binding protein or any histone-modifying enzyme. As well, thisapproach may find use in histone-specific antibody screening.

The following examples are offered to illustrate, but not to limit theclaimed invention.

EXAMPLE 1 Use of a One-Bead-One-Compound Peptide Library to IdentifyDeacetylase Specificity

General

All amino acid derivatives and resins were purchased from PeptidesInternational (Louisville, Ky.) or from Bachem (Bubendorf, Switzerland).Peptides used in the solution deacetylase assays were obtained from theUniversity of Wisconsin-Madison Biotechnology Core Facility. Otherchemical reagents were obtained from Sigma-Aldrich (St. Louis, Mo.),Acros (Geel, Belgium), Novabiochem (San Diego, Calif.), AmershamBiosciences (Buckinghampshire, England), or Quantum Dot (Hayward,Calif.). Reaction vessels for peptide library synthesis were purchasedfrom Alltech Chromatography (Deerfield, Ill.).

Analytical gradient HPLC was conducted on a Shimadzu series 2010C HPLCwith a Vydac C18 column (10 μm, 4.6×250 mm). All runs used lineargradients of 0.05% aqueous TFA and 0.02% TFA in acetonitrile. MALDI-TOFMS was performed on a Bruker REFLEX II using α-cyano-4-hydroxy-cinnamicacid as matrix. Fluorescent bead sorting was carried out on a COPASSelect (Union Biometrica, Holliston, Mass.) instrument. Fluorescencemicroscopy was done on an Olympus IX81 instrument (Tokyo, Japan)equipped with a Hamamatsu digital camera (Hamamatsu-City, Japan).

SIRT1, SIRT2, and ySir2 were expressed and purified as previouslydescribed (Borra et al., 2004, Biochemistry 43: 9877-9887; Borra et al.,2005, J. Biol. Chem. 280:17187-17195).

Prior to kinetic analysis, peptide concentrations were established byamino acid analysis (AAA) or by a coupled assay in which NAD⁺ leftoverfrom exhaustive deacetylation reactions (acetylated peptide wastypically incubated with 5-10 μM Sir2 and 80 μM NAD⁺ for 20 min) wasquantitatively converted to NADH with alcohol dehydrogenase andmonitored spectrophotometrically in real-time at 340 nm. Peptideconcentrations were obtained by subtracting the amount of NADH formedfrom the original amount of NAD⁺ used in the reaction.

Solution Deacetylation Assays

All solution phase Sir2 assays were carried out at 25° C. in 50 mMTris.HCl. Reactions were done in 50-100 μL with 0.1-1.5 μM enzyme,0.1-1.2 mM NAD⁺, 0.5-1000 μM peptide and 1 mM DTT. Reaction mixtureswere quenched with TFA to a final concentration of 1% after 5-10 min andnicotinamide levels were quantitated by HPLC at 264 nm.

Alternatively, [³²P]-NAD⁺ (10 mCi/mL) was used in assays and quenchedreaction mixtures were spotted on a silica TLC plate and run in achamber containing 60% ethanol and 40 2.5 mM ammonium acetate for 3-4hours. Levels of ³²P-OAADPr and ³²P-NAD⁺ were then quantitated byphosphorimaging and the fraction turnover was calculated. Saturationcurves were done at varying concentrations of peptide while holding thatof NAD⁺ constant. Time points were chosen such that product formationnever exceeded 20% conversion and data were plotted as rate (s⁻¹) vs.peptide concentration. Plots were fitted to the Michaelis-Mentenequation, v=[(k_(cat)/K_(m))[S]]/(1+[S]/K_(m)) using Kaleidagraphsoftware (Reading, Pa.) to extract K_(m) and k_(cat)/K_(m).

Screening Methodology

The screening strategy used the reaction of biotin N-hydroxy-succinimideester with the newly generated ε-amino group formed upon SIRT1deacetylation. Subsequent binding of the streptavidin conjugated quantumdots provided the fluorescent tag for screening.

Initially, it was established that quantum dot labeling was proportionalto the molar abundance of reacted biotin. Resin bearing free aminogroups were aliquoted into five reaction vessels and labeled with 1,0.5, 0.01, 0.001 and 0 equivalents of biotin N-hydroxy-succinimideester. After differential labeling, the resin was pooled into reactionvessel and a streptavidin conjugated quantum dot (λ_(em)=605 nm)solution was added. After draining the quantum dot solution and washingthe resin, the resulting pooled beads displayed differential levels ofassociated quantum dots, correlating with the amount of covalentlylinked biotin.

To provide a quantitative assessment of fluorescent quantum dotlabeling, a complex object parametric analyzer and sorter (COPAS)instrument was utilized. COPAS sorts beads based on fluorescenceintensity while also gathering data on bead size (time of flight). Usingthis instrument, beads labeled in the previously mentioned experimentwere sorted with an excitation of 488 nm and an emission of 610 nm. Thefluorescence distribution was plotted and distinct populations could bevisualized (FIG. 1). FIG. 1 depicts a log scale plot of fluorescenceintensity vs. TOF (bead size) for quantum dot labeled beads withbiotinylation levels of 1, 0.5, 0.01, 0.001 and 0 equivalents asanalyzed by the COPAS beadsorter at 610 nm.

These populations resided in a fluorescence regime that encompassed morethan two orders of magnitude. Moreover, the bead groupings correspondedto the differential levels of biotinylation (although the 0.001 and 0biotin equivalents coalesced into a single cluster). When the emissionwavelength was set to green light (λ_(ex)=510 nm) corresponding to theintrinsic TentaGel autofluorescence, only a single population wasobserved. Quantum dot labeling was found to be quantitative insub-stoichiometric amounts. It can be coupled to a sorting instrumentsuch as the COPAS instrument for sorting beads on the basis offluorescence intensity.

FIG. 2 depicts a microscopic representation of the differentiallybiotinylated TentaGel beads after incubation with streptavidin coatedquantum dots. Quantum dot labeled beads appear white (orange/red inoriginal) while TentaGel autofluorescence is gray (green in original).The varying shades of brightness (orange in original) correlatedqualitatively to the amount of bound quantum dot.

Library Design

Prior to library construction, it was essential to determine whetherrelatively short acetyl-peptides would function as efficient substratesof Sir2 enzymes. To evaluate peptide length requirements, 10acetyl-lysine containing peptides corresponding to the histone H3sequence surrounding Lys-14 and of varying length were assayed usingSIRT1 and a variety of other sirtuins (SIRT2; yeast Sir2, ySir2; andTrypanosoma brucei Sir2, TbSir2). Deacetylation assays were conducted atfixed NAD⁺ concentrations while peptide concentrations were varied toproduce saturation curves. The resulting data were fitted to theMichaelis-Menten equation to yield catalytic efficiencies, as defined bythe apparent second order rate constant (k_(cat)/K_(m)), which takesinto consideration both binding and catalysis. All peptides used inthese studies were N-terminally acetylated, but the N-terminus was notdeacetylated by sirtuins in control assays.

The results, shown in Table 1, are represented as relative k_(cat)/K_(m)values, with the longest peptide AcTGG(AcK)APRK (SEQ ID NO: 9) given avalue of one. In these studies, all sirtuins surveyed showed no morethan a 2 to 3-fold difference in k_(cat)/K_(m) for the various peptidesubstrates. Thus, the shortest peptide, a 5-mer, was similar incatalytic efficiency to the longest peptides in this preliminary set,regardless of the enzyme assayed. These observations suggest amino acidsbeyond the −2 and +2 positions are not necessary for efficient bindingand catalysis by sirtuins. For library construction, balancing minimalpeptide length with practical limitations of library complexity wereimportant considerations. Consequently, a 5-mer library with anacetylated lysine residue in the central position was constructed. TABLE1 Summary of the relative catalytic efficiencies (k_(cat)/K_(m)) ofvarious Sir2 homologs with ten peptide substrates Relative k_(cat)/K_(m)Peptide SIRT1 SIRT2 ySir2 TbSir2 SEQ ID NO: 3 AcGG(AcK)AP 1.72 ± 0.480.56 ± 0.07 1.51 ± 0.69 1.27 ± 0.16 SEQ ID NO: 4 AcTGG(AcK)AP 0.54 ±0.11 0.55 ± 0.07 1.62 ± 0.62 0.74 ± 0.18 SEQ ID NO: 5 AcSTGG(AcK)AP 1.61± 0.46 0.78 ± 0.09 1.83 ± 0.83 0.59 ± 0.12 SEQ ID NO: 6 AcGG(AcK)APR0.68 ± 0.12 0.60 ± 0.10 2.53 ± 1.29 1.18 ± 0.22 SEQ ID NO: 7AcTGG(AcK)APR 0.64 ± 0.10 0.72 ± 0.09 1.72 ± 0.81 0.89 ± 0.23 SEQ ID NO:8 AcSTGG(AcK)APR 0.67 ± 0.10 0.93 ± 0.20 1.60 ± 0.79 1.39 ± 0.16 SEQ IDNO: 9 AcGG(AcK)APRK 1.01 ± 0.18 0.68 ± 0.10 2.33 ± 0.93 1.82 ± 0.23 SEQID NO: 10 AcGG(AcK)APRKQ 0.80 ± 0.21 0.66 ± 0.09 2.09 ± 0.79 1.43 ± 0.20SEQ ID NO: 11 AcKSTGG(AcK)AP 0.71 ± 0.11 0.59 ± 0.10 2.12 ± 0.81 1.81 ±0.69 SEQ ID NO: 12 AcTGG(AcK)APRK 1.00 ± 0.08 1.00 ± 0.09 1.00 ± 0.361.00 ± 0.09Library Construction

After validating that quantitative quantum dot labeling could be used inconjunction with fluorescence-based bead sorting, an OBOC peptidelibrary was constructed using the split-pool method (Lam et al., 1991,Nature 354: 82-84; Furka et al., 1991, Int. J. Pept. Protein Res. 37:487-493). Eighteen variable amino acids were used at four positionscentered around an acetylated lysine (two amino acids on each side ofthe acetylated lysine residue). All common natural amino acids excludingcysteine, lysine, methionine and arginine were used. To mimic chargedresidues, dimethyl arginine was substituted for lysine and arginine. Toavoid unwanted cyanogen bromide cleavage points, isosteric norleucinewas used in place of methionine. Lysine and cysteine were not includedin the library because both residues would produce false hits (inaddition to the problems posed by disulfide formation in the lattercase) because the nucleophilicity of the amine and sulfhydryl groupsrespectively would result in their biotinylation and subsequent quantumdot labeling.

The acetylated peptide library was constructed on TentaGel Macrobead NH₂resin (280-320 μm, 0.21 mmol/g loading, 65,550 beads/g) using thesplit-pool approach. Fmoc/tBu methodology (Bodanszky, 1993, Principlesof Peptide Synthesis, 2nd ed., Springer-Verlag, Germany) was used tocarry out the library synthesis on 4.80 g of resin. Prior torandomization, a four amino acid linker, BBRM (B=β-alanine) wassynthesized. After deprotecting the N-terminus with 20% (v/v) piperidinein DMF for 15 min, the resin was split equally into eighteen separatereaction vessels (one for each amino acid in the library). Fourequivalents of amino acid and coupling reagent in addition to 5%(mol/mol) capping reagent were added to each vessel for latersequencing.

Capping reagents included phenylacetic acid and 4-pentenoic acid (FIG.3). Phenylacetic acid was used in conjunction with norleucine, while4-pentenoic acid was used with all other amino acids. Equimolar ratiosof both capping reagents were used for isoleucine, asparagine, glutamineand histidine. After a second coupling, the resin from all vessels waswashed three times each with DCM and DMF, pooled and deprotected. Next,the resin was redistributed into the reaction vessels for coupling ofthe second randomized residue. The process was repeated and afterpooling, N-ε-acetyl lysine was installed as the third residue with nocapping (FIG. 4). The split-pool technique was repeated for the fourthand fifth randomized residues. After the final N-terminal deprotection,the N-termini of all the peptides were acetylated (70% DCM, 25% aceticanhydride, 5% triethylamine) and washed with DCM. Reagent K(TFA/EDT/thioanisole/water/phenol: 82.5%, 2.5%, 5%, 5%, 5%) (King etal., Int. J. Pept. Protein Res. 36: 255-266) was used as the globaldeprotection cocktail. The resin was washed thoroughly with DCM andstored at −20° C. until use.

In preliminary studies, incorporation of arginine residues beyond thelinker position gave false positive signals in the on-bead assays, dueto reaction with the biotin ester during the labeling step. This was anunfortunate result, as it precluded incorporation of arginine in thelibrary. The same problems posed by the reactivity of arginine haveprevented its incorporation in a previous library (Hu et al., 1999,Biochemistry 38: 643-650). To mimic positively charged residues, lysineand arginine, symmetrical dimethyl arginine was used. Thus, 18⁴=104,907sequences were represented in the library. A threefold excess of beadswas used to give 95% probability that all sequences were represented(Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402).

After library synthesis, the on-bead SIRT1 deacetylation assay wascarried out, as shown in FIG. 5. In this assay, all beads weresimultaneously subjected to deacetylation conditions (0.35 μM SIRT1, 12min at 25° C.), allowing competition of all peptide sequences forreaction with SIRT1. Afterwards, the beads were washed and subjected tobiotinylation conditions in DMF. Excess reagent was removed prior toblocking non-specific protein binding sites with BSA and subsequentquantum dot labeling. Lastly, beads were washed a final time and sortedusing a COPAS instrument to obtain data such as those shown in FIG. 6.

FIG. 6 shows an example of the fluorescence distribution of librarymembers. The histogram displays the number of beads versus fluorescenceintensity of a portion of the library. Note that the sharp peak on theleft corresponds to bubbles trapped in the instrument.

FIG. 7 shows a representative mass spectrum obtained frommicrosequencing of the cleavage products of one of the top forty mostfluorescent beads (top hit sequence). The amino acids corresponding tovarious mass differences are annotated. Signature doublets are obtainedfor asparagine and histidine as result of the use of both cappingreagents (1 and 2; see FIG. 3) during those coupling reactions.

Determining the viability of quantitative quantum dot analysis was alsoperformed. Five 10 mg portions of TentaGel S NH₂ resin (90 μm, 0.26mmol/g loading, 2.86×10⁶ beads/g) were divided out and swollen in DCM.After washing with DCM (3×1 mL) and DMF (3×1 mL), the beads were labeledwith 1, 0.5, 0.01 and 0.001 molar equivalents ofN-hydroxysuccinimidobiotin in 200 μL portions of DMF. After an hour ofrocking at room temperature, the solutions were drained and washed withDMF (3×1 mL). Approximately 5 mg of resin from each of the abovereactions were combined and incubated with 1 mL BSA (1 mg/mL) in TBSTbuffer (25 mM Tris.HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1hour. The resin was washed with TBST buffer (3×1 mL) and drained to thelevel of the resin bed. At this point, 500 μL of 75 nM streptavidincoated Q-dot 605 in TBST buffer was poured over the resin and rocked for2 hours, after which the solution was drained to the resin bed beforewashing with TBST (10×1 mL). Beads were photographed using afluorescence microscope with a FITC filter and sorted on the basis offluorescence (λ_(ex)=488 nm, λ_(em)=610 nm) with a COPAS Select sortinginstrument. Sorting data were evaluated with FCS Express (De NovoSoftware, Thornhill, Ontario) in histogram and dot plot form.

On-Bead Peptide Library Deacetylation by SIRT1

The entire library was assayed in a 75-mL column equipped with a filter.Prior to the assay, the resin was sequentially washed with DCM (3×50mL), DMF (3×50 mL) and deacetylation assay buffer (50 mM Tris, pH 7.5)(1×50 mL). The enzymatic reaction was initiated upon addition of 50 mLof deacetylation cocktail (0.35 μM SIRT1, 1.5 mM β-NAD⁺, 1 mM DTT). Thereaction mixture was allowed to rock gently for 12 min. After draining,the resin was washed with doubly distilled water (5×50 mL) and DMF (5×50mL). Afterwards, the resin was rocked with biotin N-hydroxy-succinimideester in DMF (3.5 mM, 50 mL) for 20 min. The solution was drained andthe resin was washed with DMF (6×50 mL) and TBST buffer (2×50 mL).

To reduce nonspecific binding, the beads were incubated with 50 mL ofBSA (2 mg/mL) in TBST buffer for 1.5 hours. After draining and washingwith TBST buffer (1×50 mL), 50 mL of 4 nM streptavidin coated Q-Dot 605in TBST buffer was added and the mixture was allowed to rock for 2hours. Again, the solution was drained and washed with TBST buffer(10×50 mL). The resin was then suspended in a minimal amount of TBSTbuffer and refrigerated at 4° C. overnight.

Strategy for Sequencing Peptides on Beads

In order to extract peptide sequences from individual beads in thelibrary, a previously developed capping method was improved, in whichsequence decoding is done by reading a mass spectral peptide ladder(Youngquist et al., 1995, J. Am. Chem. Soc. 117: 3900-3906). Instead ofusing the acetyl group for capping during peptide synthesis, twocarboxylic acids were used: phenylacetic acid (1) and 4-pentenoic acid(2) (FIG. 3). First, a four amino acid linker was synthesized ontoTentaGel beads to extend the bound peptide into solution and to bringthe peptide mass out of the MALDI matrix region. This linker wascomposed of methionine (for a cyanogen bromide cleavage point), arginine(for improved mass spectral analysis) and two β-alanines (for addedflexibility). This capping method, which utilizes two caps, allows forthe identification of up to three isobaric (i.e.,—of identical mass)amino acids.

In each coupling step of a randomized residue, a small amount of cappingreagent was added to terminate chain growth for later sequencing (FIG.4). In each capping step, either one or both of the capping reagentswere used. The use of two reagents assisted in deciphering amino acidsof similar or identical masses. In cases, where both caps were used, asignature doublet would appear on the mass spectrum. By HPLC analysis,it was determined that 5 mol % capping at each step in the synthesis ofa prototypical 5-mer yielded ˜79% full-length peptide. This amount ofcapping reagent provided a more than adequate amount for on-bead assay,yet produced enough capped material to produce quality peptide laddersin the mass spectra. An acetyl group served as the N-terminal cap.

Library Screening

Beads were sorted on the basis of fluorescence (λ_(ex)=488 nm,λ_(em)=610 nm) using the COPAS instrument. Initially, the 300 mostintensely fluorescent beads (0.1%) were collected, pooled and thensorted a second time to generate an enriched sample of the 45 brightestbeads. After washing in a guanidinium hydrochloride solution, singlebeads were placed in separate microcentrifuge tubes and treatedovernight with a cyanogen bromide cleavage cocktail. The cleavageproducts were subsequently subjected to MALDI-TOF MS for sequenceanalysis (FIG. 7). Of those 45 beads, 33 were sequenced successfullyfrom their mass spectra (Table 2), 6 were found to be damaged and werenot sequenced, while the remaining 6 yielded spectra that were notinterpretable. BLAST searches of the mammalian proteome were performedin the short, nearly exact mode for the 33 sequences obtained from thelibrary (see Table 4).

Shown in Table 2 are peptide sequences of hits from the SIRT1combinatorial library screen. Position −2 is the N-terminal end andPosition +2 is the C-terminal end. Uncertainty in the order ofN-terminal (and adjacent) amino acids is signified by the symbol /.TABLE 2 Peptide sequences of hits from the SIRT1 combinatorial libraryscreen Position −2 Position −1 Position 0 Position +1 Position +2 LeuAsn AcLys Asp Gln Trp His AcLys Phe Gln Trp His AcLys Phe Glu Ser TyrAcLys Gln Trp Gln Pro AcLys Gln Ile Val Gln AcLys Ile Ile His Me₂ArgAcLys Nle Pro Ala Val AcLys Phe Nle Asn His AcLys Leu Leu Me₂Arg PheAcLys Pro Glu Nle Nle AcLys Gln Gln Trp Gly AcLys Ser Pro Phe Glu AcLysTyr Me₂Arg Trp Pro AcLys Trp Gln Me₂Arg Ala AcLys Nle Asp Gly Thr AcLysThr Gly Gly Tyr AcLys Pro Thr Ile Phe AcLys Thr Phe Thr Glu AcLys GlnGlu His Trp AcLys Thr His Asp Ser AcLys Gly Ala Ser Asp AcLys Tyr HisAsn His AcLys Ile Ile Trp Trp AcLys His Gly Pro Ile AcLys Glu Gln Me₂ArgPro AcLys Gln Phe Asp Val AcLys Nle His Ile Tyr AcLys Asn Asp Thr ProAcLys Asn Ala Pro Gly AcLys Leu Tyr Me₂Arg/Trp Me₂Arg/Trp AcLys Ile ThrPro/Trp Pro/Trp AcLys Ile Thr Me₂Arg/Pro Me₂Arg/Pro AcLys Ser IleHit Sequencing with MALDI-MS

Beads from the enriched sample were pooled and washed with 8 Mguanidinium hydrochloride (2×1 mL), doubly distilled water (10×1 mL) andDMF (3×1 mL). Individual beads were then deposited into separatemicrocentrifuge tubes containing 20 μL of cleavage cocktail (70% TFA,30% doubly distilled water and 20% cyanogen bromide by weight), asdescribed by Hu et al., 1999). After incubation overnight in the dark,the samples were dried and resuspended in 5 μL of 0.1% TFA. Each sample(1 μL) was combined with saturated matrix solution (1 μL) and dried onthe target for MALDI-TOF MS analysis (positive ion mode).

Library Validation

To validate the results of the library screen, select hits and non-hitswere resynthesized and subjected to in-solution kinetic analysis (Table3). A radioactive TLC-based assay was employed with subsaturating levelsof [³²P]-NAD⁺ to determine the relative catalytic efficiencies (Jacksonet al., 2003, J. Biol. Chem. 278: 50985-50998). In addition, two“consensus” peptides containing residues occurring with the highest andlowest frequency at each position, independent of context, wereanalyzed. For comparison, a 5-mer comprised of a sequence correspondingto a known site for p53 deacetylation by SIRT1 was assayed (Table 3).

In Table 3, efficiencies (^(x)=average) were obtained by fitting thedata from [³²P]-NAD⁺ assays to the modified Michaelis-Menten equation,v=[(k_(cat)/K_(m))[S]]/(1+[S]/K_(m)). No definite catalytic efficiencyfor VQ(AcK)II was established due to problems with insolubility; a lowerlimit was established. Catalytic efficiencies of peptides containing theresidues of the highest^(b)/lowest^(c) frequency at each position andthe sequence relevant to p53 deacetylation in vivo^(d) are shown forcomparison. TABLE 3 Peptide sequences and catalytic efficiencies ofresynthesized select hits and non-hits from the SIRT1 peptide libraryscreen k_(cat)/K_(m) Peptide (× 10⁻³ M⁻¹s⁻¹) Select Hits SEQ ID NO: 13QP(AcK)QI 27.2 ± 4.2 SEQ ID NO: 14 Me₂RP(AcK)QF 14.7 ± 3.2 SEQ ID NO: 15Me₂RP(AcK)SI 8.36 ± 0.57 SEQ ID NO: 16 NH(AcK)lI 3.63 ± 0.80 SEQ ID NO:17 WH(AcK)FQ 3.29 ± 0.43^(x) SEQ ID NO: 18 VQ(AcK)II^(a) ≧2.47 ±1.27^(x) Select Non-hits SEQ ID NO: 19 AY(AcK)EV 5.32 ± 0.63 SEQ ID NO:20 QNIe(AcK)GF 2.37 ± 0.14 SEQ ID NO: 21 LNIe((AcK)VG 1.61 ± 0.48^(x)For Comparison SEQ ID NO: 22 WH(AcK)QQ^(b) 7.23 ± 1.12 SEQ ID NO: 23WP(AcK)QQ^(b) 1.54 ± 1.06 SEQ ID NO: 24 EL(AcK)AS^(c) 1.39 ± 0.10 SEQ IDNO: 25 HK(AcK)LM^(d) 3.11 ± 0.45

Hit sequences had significantly higher catalytic activity than non-hits.Some hits were near or greater than an order of magnitude morecatalytically active than their non-hit counterparts. Hits correlatedwith increased catalytic activity by as much as 20-fold. Most hitsequences assayed in solution had significantly higher activity than thepeptide sequence relevant to in vivo p53 deacetylation. One non-hitsequence AY(AcK)EV had a catalytic activity comparable to those of a fewof the hits.

Although the apparent second order rate constant (k_(cat)/K_(m)) variedwidely among the peptides tested, the turnover number (k_(cat)) wasrelatively constant at ˜0.1 s⁻¹. Differences in k_(cat)/k_(m) reflectdifferences in peptide binding affinity.

One of the main advantages of this OBOC library is its context-specificnature. In other words, there is no implicit assumption that residues insubstrate sequences function independently of one another. Whileoriented peptide libraries can be useful in resolving globally-preferred“consensus” sequences (Songyang et al., 1994; Blander et al., 2005) theydo not provide contextual information.

The so-called “consensus” peptides WH(AcK)QQ and WP(AcK)QQ show aseven-fold difference in catalytic activity in favor of WH(AcK)QQ (Table3). Thus, in the context of WX(AcK)QQ, a histidine is greatly preferredat position −1. Within the XP(AcK)QX context, QP(AcK)QI is favored overWP(AcK)QQ by 18-fold. Thus, SIRT1 mediated deacetylation is stringentlycontext dependent and that there is no best “average sequence”. Furthersupport comes from the fact that although proline residues (at −1) arenot well tolerated when adjacent to a tryptophan at −2, they appear tofunction well when adjacent to dimethyl arginine at −2. There aresynergistic/antagonistic relationships among certain residues and thatthis plays a significant role in substrate recognition by SIRT1.

BLAST searches of the SIRT1 hits (Table 2) within the mammalian proteomereveal correspondence to a number of proteins (Table 4), some of whichare known to be acetylated in vivo. TABLE 4 BLAST searches of SIRT1 hitsequences Hit Se- Protein (name, accession, Sequence ID quence relevantsequence) SEQ ID NO: 26 LNKDQ Moesin, NP_002435, [Homo sapiens] MSNprotein, AAH11827 [Homo sapiens] SEQ ID NO: 27 WHKFQ chondroitin sulfateproteogly- can, 2 NP_004376, [Homo sapiens] SEQ ID NO: 28 WHKFE dualoxidase 1 precursor NP_059130 [Homo sapiens] NADPH thyroid oxidase 2,AAF73922, [Homo sapiens] SEQ ID NO: 29 SYKQW fatty acid coenzyme Aligase 5, BAA86054 [Homo sapiens] SEQ ID NO: 30 QPKQI Notch homolog 4(Drosophila), CA117543 [Homo sapiens] Orphan sodium- and chloride-dependent neurotransmitter transporter, Q9GZN6, [Homo sapiens] SLC6A16protein, AAH34948 [Homo sapiens] TPA: class II bHLH protein scleraxis,DAA00239 [Homo sapiens] U5 snRNP-specific protein, AAH64370 [Homosapiens] apoptosis-regulated protein 1, AAS64748 [Homo sapiens] SEQ IDNO: 31 VQKII Werner syndrome, AAR05448 [Homo sapiens] chaperonincontaining TCP1, subunit 6B, NP_006575 [Homo sapiens] MGC16733 protein,AAH09995 [Homo sapiens] chemokine (C-X-C motif) ligand 3, NP_002081[Homo sapiens] MDN1 protein, AAH14882 [Homo sapiens] LOC150159 protein,AAH46636 [Homo sapiens] C9orf72 protein, AAH68445 [Homo sapiens] SEQ IDNO: 32 HRKMP SCAM-1 protein, AAH67260 [Homo sapiens] SH3-containingadaptor mole- cule-1, AAC09244 [Homo sapiens] KIAA1792 protein, BAB47421[Homo sapiens] RP5-1187M17.5, CAC32460 [Homo sapiens] MSTP060, AAO15306[Homo sapiens] LAS1-like, NP_112483 [Homo sapiens] OTTHUMP00000021323,CAH70992 [Homo sapiens] SEQ ID NO: 33 HKKMP FLJ00158 protein, BAB84913[Homo sapiens] SEQ ID NO: 34 AVKFM secreted frizzled-related pro- tein5, CAI14274 [Homo sapiens] RAN binding protein 17 [Homo sapiens] SEQ IDNO: 35 NHKLL protocadherin 11, AAK13468 [Homo sapiens] engulfment andcell motility 3, NP_078988 [Homo sapiens] PARP8 protein, AAH37386 [Homosapiens] complement component 3, AAR89906 [Homo sapiens] WD repeatdomain 35, isoform 2, AAH36659 [Homo sapiens] SEQ ID NO: 36 RFKPE solutecarrier family 30 (zinc transporter), NP_037441 member 4 [Homo sapiens]ubiquitin specific protease 53, NP_061923 XP_052597 [Homo sapiens]membrane-associated guanylate kinase-related 3 (MAGI-3), CAH70944 [Homosapiens] zinc transporter 4, AAB82561 [Homo sapiens] KIAA1350 protein,BAA92588 [Homo sapiens] cytochrome P450, family 2, sub- family E,polypeptide 1, NP_000764 [Homo sapiens] SEQ ID NO: 37 KFKPE Pleckstrinhomology domain con- taining, family A (phosphoino- sitide bindingspecific) member 3, AAH44567 [Homo sapiens] Phosphoinositol 4-phosphateAdaptor Protein-1, AAG15199 [Homo sapiens] SEQ ID NO: 38 MMKQQ golgiantigen gcp372, BAA05025 [Homo sapiens] giantin, CAA53052 [Homo sapiens]SEQ ID NO: 39 WGKSP apolipoprotein L5, NP_085145 [Homo sapiens]NY-REN-55 antigen, AAD42879 [Homo sapiens] NIMA (never in mitosis genea)- related kinase 1, NP_036356 XP_291107 [Homo sapiens] KIAA1901protein, BAB67794 [Homo sapiens] UDP-N-acetylglucosamine: alpha-1,3-D-mannoside beta-1,4-N- acetylglucosaminyltransferase IV, NP_080519[Mus musculus] SEQ ID NO: 40 FEKYR protocadherin gamma A11, AAD43765[Homo sapiens] signal-induced proliferation- associated 1 like 1,NP_056371 [Homo sapiens] high-risk human papilloma viruses E6oncoproteins tar- geted protein E6TP1 alpha; putative GAP protein alpha,AAD12543 [Homo sapiens] KIAA0440, BAA23712 [Homo sapiens] spa-1-like;similar to AF026504 (PID:g2555183), AAC83179 [Homo sapiens] PRO0097,AAF24015 [Homo sapiens] SEQ ID NO: 41 FEKYK Nebulin, P20929 [Homosapiens] SEQ ID NO: 42 WPKWQ No identical sequence match SEQ ID NO: 43RAKMD large tumor suppressor 1, AAD16882 [Homo sapiens] LATS, largetumor suppressor, homolog 2, NP_055387 [Homo sapiens] amyloidprecursor-like protein 1, AAB50173 [Homo sapiens] potassium channel,subfamily T, member 1, NP_065873 XP_029962 [Homo sapiens] LOH12CR1,AAK71328 [Homo sapiens] olfactomedin 1, AAP35810 [Homo sapiens] Zincfinger protein 541, AAI01053 [Homo sapiens] p33, AAG11396 [Homo sapiens]nucleobindin 1, AAP88830 [Homo sapiens] SEQ ID NO: 44 KAKMD meltransforming oncogene, NP_005361 [Homo sapiens] T cell receptor betavariable 21/OR9-2, CAH69869 [Homo sapiens] SEQ ID NO: 45 GTKTG WD repeatdomain 3, CA122739 [Homo sapiens] cytoplasmic linker 2 isoform 1,NP_003379 [Homo sapiens] KIAA1858 protein, BAB47487 [Homo sapiens]exophilin 5, AAM44402 [Homo sapiens] SEQ ID NO: 46 GYKPT complementcomponent 4 binding protein, alpha, CAH70782 [Homo sapiens] KCRM_HUMAN;M-CK, AAC62841 [Homo sapiens] creatine kinase, muscle, AAP35439 [Homosapiens] SEQ ID NO: 47 IFKTF cullin 4B, AAR13073 [Homo sapiens] KIAA0695protein, BAA31670 [Homo sapiens] SEQ ID NO: 48 TEKQE caspase recruitmentdomain family, member 11, EAL23962 [Homo sapiens] CARD-containing MAGUKprotein CARMA1, AAL34460 [Homo sapiens] oligophrenin-1 like protein,AAD39482 [Homo sapiens] GTPase regulator associated with the focaladhesion kinase pp125, NP_055886 [Homo sapiens] myosin phosphatase-Rhointer- acting protein, AAQ63176 [Homo sapiens] HLC-8, AAO25513 [Homosapiens] cardiomyopathy associated pro- tein 1, AAQ64003 [Homo sapiens]TRAF family member-associated Nf-kappa B activator NP_665731 [Rattusnorvegicus] RCSD1 protein, AAH98426 [Homo sapiens] SEQ ID NO: 49 HWKTHNo identical sequence match SEQ ID NO: 50 DSKGA dentinsialophosphoprotein pre- proprotein, NP_055023 [Homo sapiens]Monoglyceride lipase, AAH00551 [Homo sapiens] EGF domain-containingprotein, AAP35084 [Homo sapiens] Protein phosphatase 1, regula- tory(inhibitor) subunit 1A, AAH22470 [Homo sapiens] RNA binding motifprotein 19, NP_057280 [Homo sapiens] MEGF8, BAA32469 [Homo sapiens] zincfinger protein 608, NP_065798 XP_114432 [Homo sapiens]valyl-tRNA-synthetase G7a/Bat6, AAL14460 [Mus musculus] SEQ ID NO: 51SDKYH Deoxycytidylate deaminase, P32321 (dCMP deaminase) SEQ ID NO: 52NHKII zinc finger protein 588, NP_057304 [Homo sapiens] zinc fingerprotein 15-like 1 (KOX 8), NP_067092 [Homo sapiens] UDP-Gal:betaGlcNAcbeta 1,4- galactosyltransferase 6 var- iant, BAD92431 [Homo sapiens]SMAP-7,BAB20272 [Homo sapiens] SEQ ID NO: 53 WWKHG No identical sequencematches SEQ ID NO: 54 PIKEQ procollagen, type XII, alpha 1, NP_031756[Mus musculus] SEQ ID NO: 55 RPKQF RNA guanylyltransferase, AAB88903[Mus musculus] KIAA0992 protein, BAA76836 [Homo sapiensN-methylpurine-DNA glycosylase isoform, aNP_002425 [Homo sapiens] SEQ IDNO: 56 KPKQF RP11-334P12.2, CAH71251 [Homo sapiens] cell adhesion kinasebeta, AAC05330 [Homo sapiens] RNA binding motif protein 7, AAH34381[Homo sapiens] PTK2B protein tyrosine kinase 2 beta isoform a, NP_775266[Homo sapiens] Neuronal amiloride-sensitive cation channel 1, isoform 2,AAH75043 [Homo sapiens] RNA helicase, AAD19826 [Homo sapiens] focaladhesion kinase, AAB47217 [Homo sapiens] SEQ ID NO: 57 DVKMH Noidentical sequence matches SEQ ID NO: 58 IYKND immunoglobulinsuperfamily re- ceptor translocation associated 2(IRTA2), CAH71429 [Homosapiens] ROS1, AAA60277 [Homo sapiens] G protein-coupled receptor 119,AAP72132 [Mus musculus] transmembrane protein kinase 3), AAA36580 [Homosapiens] Fc receptor-like protein 5, AAK93971 [Homo sapiens] v-ros UR2sarcoma virus onco- gene homolog 1 (avian), CA142375 [Homo sapiens] SEQID NO: 59 TPKNA zinc finger protein 440 like, NP_001012771 XP_371138[Homo sapiens] adenosine deaminase, RNA- specific, B2, NP_443209 [Musmusculus] SEQ ID NO: 60 PGKLY Phosphatidylethanolamine binding protein,AAH08169 [Mus musculus] SEQ ID NO: 61 RWKIT integrin beta 4, NP_037312[Rattus norvegicus] SEQ ID NO: 62 KWKIT desmoglein 2, NP_031909 [Musmusculus] SEQ ID NO: 63 WRKIT chromosome 6 open reading frame 103,CAI16490 [Homo sapiens] SEQ ID NO: 64 WKKIT No identical sequence matchSEQ ID NO: 65 WPKIT EPB41L5 protein, AAH32822 [Homo sapiens] erythrocytemembrane protein band 4.1 like 4B isoform 1, NP_060894 [Homo sapiens]EHM2, BAA96079 [Homo sapiens] KIAA1548 protein, BAB13374 [Homo sapiens]beta-crystallin, AAA52107 [Homo sapiens] SEQ ID NO: 66 PWKIT REPS1protein, AAH21211 [Homo sapiens] RALBP1 associated Eps domain containing1, CAI42879 [Homo sapiens] sodium potassium chloride co- transporter 2,NP_000329 [Homo sapiens] SEQ ID NO: 67 RPKSIalpha(1,3)-fucosyltransferase; ELFT, AAB20349 [Homo sapiens] nuclearfactor I, AAB52369 [Homo sapiens] fucosyltransferase 4, NP_002024 [Homosapiens] ELAM-1 ligand fucosyltransfer- ase, AAA63172 [Homo sapiens]calmodulin regulated spectrin- associated protein 1-like 1, NP_982284XP_036589 [Homo sapiens] CAMSAP1L1 protein, AAH11385 [Homo sapiens] SEQID NO: 68 KPKSI Transient receptor potential cation channel subfamily Mmember 7 (Long transient recep- tor potential channel 7) (LTrpC7)(Channel-kinase 1), Q96QT4 [Homo sapiens] Synapse-associated protein102, AAH93864 [Homo sapiens] C21orf7 form C, AAF81753 [Homo sapiens]c21orf7 form B, AAF81752 [Homo sapiens] TAK1-like protein, AA016519[Homo sapiens] MAP4K3, AAN75849 [Homo sapiens] channel-kinase 1,AAK19738 [Homo sapiens] transcription factor GATA-6, NP_999493 [Susscrofa] SEQ ID NO: 69 PRKSI poly (ADP-ribose) polymerase family, member6 isoform 1, NP_064598 [Homo sapiens] C19orf2 protein, AAH14933 [Homosapiens] RPB5-mediating protein, isoform b, AAH67259 [Homo sapiens]NNX3, AAD08679 [Homo sapiens] SEQ ID NO: 70 PKKSI zinc finger protein318, CAH71374 [Homo sapiens] Alanyl-tRNA synthetase, AAH11451 [Homosapiens] histone H1, AAN06703 [Homo sapiens] nucleoporin, BAB18537 [Homosapiens] Inner membrane protein, mito- chondrial, AAH02412 [Homosapiens] ZNF318 protein, AAH30687 [Homo sapiens]

EXAMPLE 2 Use of a Combinatorial Library to Identify Histone-SpecificProtein Binding

General

An OBOC histone H4 N-terminal tail combinatorial library was constructedto identify the binding preferences of the antibody toward all knownpossible histone modification states. The H4 histone tail library wascomprised of the sequence corresponding to the first 21 amino acids ofhuman histone H4 attached to a linker composed of 2 β-alanines (B) and amethionine (M). The library included 800 unique peptide sequences,representing all known modification states for the first 21 amino acidsof histone H4 in addition to all possible methylation states at lysinesand arginines that are known to be methylated. Using an α-phos (S1) H4antibody as a primary antibody, the library was screened to determinehistone H4 N-terminal sequences to which the primary antibodyspecifically bound.

Amino acid derivatives and resins were purchased from PeptidesInternational (Louisville, Ky.), Novabiochem (San Diego, Calif.), orfrom Bachem (Bubendorf, Switzerland). Other chemical reagents wereobtained from Sigma-Aldrich, Invitrogen (Carlsbad, Calif.), or JacksonImmunoResearch Laboratories (West Grove, Pa.). The α-phos (S1) H4antibody was a gift from the laboratory of C. David Allis (RockefellerUniversity, New York, N.Y.). Peptides were synthesized on a Symphonysynthesizer from Protein Technologies (Tucson, Ariz.). Filter columnsfor on-bead assays were obtained from Alltech (Deerfield, Ill.).

Analytical gradient HPLC was performed on a Shimadzu series 2010C HPLCwith a Vydac C18 column (10 μm, 4.6×250 mm). All runs employed lineargradients of 0.05% aqueous TFA and 0.02% TFA in acetonitrile.Microextraction tips for desalting peptides were purchased from Varian,Inc. (Palo Alto, Calif.). MALDI-TOF MS was performed on a Bruker REFLEXII and MALDI TOF-TOF MS was executed on an Applied Biosystems 4800. AZeiss Axioplan 2 microscope (Jena, Germany) with a DAPI dye bandpassfilter (390-410 nm) and an AxioCam MRm was used for fluorescencemicroscopy.

On-Bead Assay with Peptide Standards

Five-mg quantities of TentaGel Macrobead NH₂ resin (280-320 μm, 0.27mmol/g loading, 65,550 beads/g) bearing either a phosphorylated orunphosphorylated histone H4 sequence (or a mixture) were added to 1.5 mLfilter columns, washed thoroughly with DCM, MeOH, doubly distilled water(ddH₂O) and PBST buffer (25 mM NaPi, pH 7.4, 150 mM NaCl, and 0.1% Tween20). The resin was swelled for 1 hour with gentle rocking prior todrainage and one hour of blocking with 3% (w/v) bovine serum albumin(BSA) in PBST. After draining the blocking solution to the resin bed,100 μL of a 100:1 dilution of α-phos (S1) H4 antibody in PBST containing3% BSA was added and the resin was allowed to rock gently for one hour.

After draining to the resin bed, the resin was washed 3×100 μL PBST and100 μL of 50 nM biotinylated goat-anti-rabbit antibody in PBSTcontaining 3% BSA was added. One hour of gentle rocking was followed bydraining the solution to the resin bed and washing 3×100 μL PBST.

The resin was incubated with 100 μL of 25 nM solution of Q-dot 605streptavidin conjugate in PBST and gently rocked for 2 hours. Followingdrainage to the resin bed, the resin was washed 10×200 μL PBST. Theresin was then resuspended in PBST and viewed under a fluorescencemicroscope.

Library Construction

The combinatorial histone H4 peptide library was constructed on TentaGelMacrobead NH₂ resin (280-320 μm, 0.27 mmol/g loading, 65,550 beads/g)using the split-pool approach (Lam et al., 1991, Nature 354: 82-84;Furka et al., 1991, Int. J. Pept. Protein Res. 37: 487-493) for sites ofvariability. Sites of variability include positions 20 (K, AcK, MeK,Me₂K, Me₃K), 16 (K,AcK), 12 (K,AcK), 8 (K,AcK), 5 (K,AcK), 3 (R, MeR,Me₂R_(symmetric),Me₂R_(asymmetric),citrulline) and 1 (S, pS). Thesynthesis was performed on a 50 μmol scale with standard Fmoc/tBuchemistry (Bodanszky M., 1993, Principles of Peptide Synthesis, 2nd ed.,Springer-Verlag, Germany). All amino acids (at least 4.7equivalents/coupling) were double coupled for 2 hour time periods.

Prior to the partially randomized histone H4 sequence, a 3 amino acidlinker, BBM (where B=β-alanine, M=methionine) was synthesized. After thefinal N-terminal deprotection, the N-termini of all the peptides wereacetylated with acetic anhdyride. A 50 mg (13.5 μmol) portion of thelibrary was deprotected for 5 hours with Reagent K(TFA/EDT/thioanisole/water/phenol: 82.5%, 2.5%, 5%, 5%, 5%) prior touse, as described by King et al., 1990, Int. J. Pept Protein Res. 36:255-266. The remainder of the library was stored at 4° C. for later use.

Evaluation of Integrity of the Peptide Library

Twenty beads were randomly selected from the library and deposited intoseparate microcentrifuge tubes containing 20 μL of cleavage cocktail(70% TFA, 30% ddH₂O and 20% cyanogen bromide by weight; Hu et al., 1999,Biochemistry 38: 643-650). After incubation overnight in the dark, thesamples were dried.

Ten of the cleavage products were desalted and sequenced by MALDITOF-TOF MS. The remaining 10 cleavage products were dissolved in 100 μLquantities of ddH₂O and analyzed by analytical RP-HPLC. Fractionscorresponding to the primary peak at 214 nm were lyophilized andresuspended in 5 μL of ddH₂O. Each sample (1 μL) was combined withsaturated matrix solution (1 μL) and dried on the target for MALDI-TOFMS analysis (positive ion mode).

On-Bead Library Prescreen and Screen

Prescreen was performed by adding 50 mg (13.5 μmol) of the peptidelibrary to a 4 mL filter column and washing it thoroughly with DCM,MeOH, ddH₂O and PBST buffer. The resin was swelled for 1 hour withgentle rocking prior to drainage and one hour of blocking with 3% BSA inPBST. After draining the blocking solution to the resin bed, 1 mL of 50nM biotinylated goat-anti-rabbit antibody in PBST containing 3% BSA wasadded. Following 1 hour of rocking, the solution was drained to theresin bed and washed 3×1 mL PBST. The resin was then rocked with 1 mL ofa 25 nM solution of Q-dot 605 streptavidin conjugate in PBST for twohours. Following drainage to the resin bed, the resin was washed 10×2 mLPBST. At this point, the resin was resuspended in PBST and viewed undera fluorescent microscope and any fluorescent beads could be removed fromthe library.

After prescreening the library for nonspecific interactions with thesecondary antibody or the quantum dots, a screen was performed. The onlydifference from the prescreen was a one hour incubation with 1 mL of a100:1 dilution of α-phos (S1) H4 in PBST with 3% BSA after the swellstep and washing 3×1 mL PBST prior to addition of the secondaryantibody. When viewed under the microscope, a number of brightlyfluorescent, moderately fluorescent and dark beads were manuallyselected.

Peptide Sequencing with MALDI TOF-TOF MS

Beads that were selected under the microscope were incubated with 200 μLof 8 M guanidinium hydrochloride prior to washing 3×500 μL ddH₂O anddrying. Peptides were cyanogen bromide cleaved from each bead anddesalted before sequencing with MALDI TOF-TOF MS.

On-Bead Western Analysis

FIG. 8 shows schematically the on-bead Western (immunoprecipitation)analysis with control peptides. FIG. 8 shows the on-bead assay, beadswith phosphorylated sequences (top; phosphorylation depicted as grayovals attached to peptide chains) or unphosphorylated sequences (bottom;naked peptide chains) corresponding to the N-terminal tails of histoneH4 are first incubated with α-phos (S1) H4 antibody. After a washingstep, a biotinylated secondary antibody directed towards the primaryantibody is added. After another washing step, streptavidin-coatedquantum dots are incubated with the beads.

FIG. 9 shows: (left panel) fluorescent microscopic image ofAcSGRGKGG(AcK)GLG(AcK)GGAKRHRKVBBM-Macrobead (1) (SEQ ID NO:1) after theon-bead assay; (center panel) a fluorescent microscopic image ofAcpSGRGKGG(AcK)GLG(AcK)GGAKRHRKVBBM-Macrobead (2) (SEQ ID NO:2); (rightpanel) a fluorescent microscopic image of a 5:1 ratio of (1) to (2).B=beta-alanine.

After demonstrating detection of serine phosphorylation of a histone H4sequence immobilized on a bead (FIG. 9), an OBOC histone H4combinatorial library (H4 histone N-terminal tail library) wasconstructed to further examine the binding preferences of the antibodytoward all known possible histone modification states. The H4 histonetail library, schematically shown in FIG. 10, is comprised of thesequence corresponding to the first 21 amino acids of human histone H4attached to a linker composed of 2 β-alanines (B) and a methionine (M).Sites for modification include positions 20, 16, 12, 8, 5, 3 and 1 andare annotated with X. Possible modification states are shown above orbelow the peptide chain (FIG. 10). In this library, schematically shownin FIG. 10, the first 21 amino acids of histone H4 are represented with7 sites of variability. The sites include positions 20 (K, AcK, MeK,Me₂K, Me₃K), 16 (K,AcK), 12 (K,AcK), 8 (K,AcK), 5 (K,AcK), 3 (R, MeR,Me₂R_(symmetric),Me₂R_(asymmetric),citrulline) and 1 (S, pS). Thislibrary includes all known modification states for the first 21 aminoacids of histone H4 in addition to all possible methylation states atlysines and arginines that are known to be methylated. Therefore, thislibrary is composed of 800 unique peptide sequences with 99% confidenceof 95% coverage of combinatorial space (each library consists of −50 mgof resin; Burgess et al., J. Med. Chem. 37: 2985-2987).

The library synthesis was followed by rigorous evaluation of thesynthetic product. RP-HPLC analysis of the cleavage products from 10individual beads revealed peptides of −90-95% purity within the correctmass range. In addition, the cleavage products from 10 additionalrandomly selected beads were successfully sequenced with MALDI TOF-TOFMS.

The library was first prescreened with only the secondary antibody andquantum dots. The fact that none of the beads exhibited fluorescence dueto quantum dots suggested the absence of non-specific interactionsbetween the immobilized peptides with either the secondary antibody orthe quantum dots. Therefore, when the primary antibody was included in ascreening experiment, the fluorescence observed was due to a specificinteraction with the primary antibody (FIG. 11). Of the library, abouthalf of the beads exhibited some level of quantum dot-associatedfluorescence. A number of individual beads of were manually selected andclassified as either: fluorescent, moderately fluorescent or dark.

FIG. 11 shows a fluorescent microscopic image of the results of a H4library screen with α-phos (S1) H4 antibody, which was used as a primaryantibody. The fluorescence intensity is indicative of the degree ofinteraction of peptides with the α-phos (S1) H4 antibody. A number ofbeads were manually selected for peptide sequencing based on theirfluorescence intensity.

Data from the screen indicate the binding preferences of the α-phos (S1)H4 antibody for certain sequences (Table 5). Twenty beads were manuallyselected from a screen of a histone H4 tail library. Sequences wereelucidated by MALDI TOF-TOF MS. All sequences obtained from fluorescentbeads were phosphorylated while the moderately fluorescent beadsdisplayed peptides that were typically phosphorylated (and generallyhighly-modified). Eighty percent of the dark beads harbored peptidesthat were unphosphorylated. Legend: pS=phosphorylated serine,AcK=acetylated lysine, MeK, Me₂K, Me₃K=the correspondingly methylatedstates of lysine, MeR and Me₂R=the correspondingly methylated states ofarginine where (a) and (s) refer to symmetric and asymmetricrespectively. TABLE 5 Binding preferences of the α-phos (S1) H4 antibodyBead Position Position Position Fluorescence Position 1 Position 3Position 5 Position 8 12 16 20 Fluorescent pS Me₂R (a) AcK K K AcK MeKFluorescent pS Me₂R (a) K/AcK K/AcK K K MeK Fluorescent pS Me₂R (a) KAcK MeK K AcK Fluorescent pS MeR AcK AcK K K AcK/Me₃K Fluorescent pS R KAcK Me₂K K MeK Fluorescent pS MeR K AcK K K AcK/Me₃K Fluorescent pS R KK AcK/Me₃K K MeK Fluorescent Poor quality (3 beads) Moderately pS MeRAcK AcK Ack/Me₃K AcK Me₂K Fluorescent Moderately pS Me₂R (s) AcK AcK MeKAcK Me₂K Fluorescent Moderately S Me₂R (s) AcK AcK Me2K K K FluorescentModerately Poor quality (but appear to be phosphorylated and heavilymodified - 2 beads) Fluorescent Dark S Me₂R (s) K K MeK K K Dark S R K KMe₂K K AcK/Me₃K Dark pS Me₂R (s) K AcK AcK/Me₃K AcK MeK Dark S MeR K KAcK K MeK Dark S MeR K K Me₂K K Me₃K

It is to be understood that this invention is not limited to theparticular devices, methodology, protocols, subjects, or reagentsdescribed, and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which is limited only by the claims. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered in clinical prevention and therapy, obvious tothose skilled in the art, are within the scope of this invention. Allpublications, patents, and patent applications cited herein areincorporated by reference in their entirety for all purposes.

1. A method for determining binding specificity of a protein deacetylase, comprising: a) contacting the protein deacetylase with a combinatorial acetyl-peptide library, wherein the peptide library comprises a plurality of solid phase supports linked to a different and distinct acetyl-peptide; b) labeling deacetylated peptides with a label specific for deacetylated peptides; and c) correlating the label intensity with peptides that are deacetylated by the protein deacetylase, thereby determining binding specificity of the protein deacetylase.
 2. The method of claim 1 wherein the label is specific for an amino group formed upon deacetylation.
 3. The method of claim 1 wherein the solid phase supports are beads.
 4. The method of claim 1 wherein the label is colorimetric, radioactive, or fluorescent.
 5. The method of claim 1 wherein the label is a labeled quantum dot.
 6. The method of claim 1 further comprising sorting the solid phase supports from the labeled peptide library on the basis of label intensity.
 7. The method of claim 1 further comprising determining the sequence of the peptide attached to the solid phase support.
 8. The method of claim 1 wherein each peptide sequence comprises at least 5 amino acids.
 9. The method of claim 1 wherein the protein deacetylase is a sirtuin.
 10. The method of claim 1 wherein the protein deacetylase is SIRT1.
 11. The method of claim 8 wherein the peptide sequence is selected from the group consisting of: LNKDQ, WHKFQ, WHKFE, SYKQW, QPKQI, VQKII, HRKMP, HKKMP, AVKFM, NHKLL, RFKPE, KFKPE, FEKYR, MMKQQ, WGKSP FEKYK, WPKWQ, RAKMD, KAKMD, GTKTG, GYKPT, IFKTF, TEKQE, HWKTH, DSKGA, SDKYH, NHKII, WWKHG, PIKEQ, RPKQF, KPKQF, DVKMH, IYKND, TPKNA, PGKLY, RWKIT, KWKIT, WRKIT, WKKIT, WPKITPWKIT, RPKSI, KPKSI, PRKSI, and PKKSI.
 12. A method for determining binding specificity of an enzyme, comprising: a) contacting a combinatorial peptide library with the enzyme, wherein the peptide library comprises a plurality of solid phase supports, wherein each solid phase support is linked to a different and distinct peptide; b) labeling the peptides with a label specific for peptides covalently modified by the enzyme; and c) correlating the intensity of the label with peptides that are covalently modified by the enzyme, thereby determining binding specificity of the enzyme.
 13. The method of claim 12 wherein the covalent modification comprises post-translational modification.
 14. An analytical method comprising: a) generating a combinatorial library of peptides comprising one or more peptide sequences attached to a solid phase support, wherein each peptide comprises two or more chemically modified amino acids, wherein the combinatorial library comprises a plurality of solid phase supports linked to a different and distinct peptide; b) contacting the combinatorial library with a protein; c) detecting the protein bound to one or more peptides using a label; and d) correlating the label intensity with peptides to which the protein binds, thereby determining the binding specificity of the protein.
 15. The method of claim 14 wherein detecting the protein bound to the one or more peptides is performed with a complex comprising a compound specific for the protein and a detectable label.
 16. The method of claim 14 wherein the solid phase supports are beads.
 17. The method of claim 14 wherein the protein is labeled.
 18. The method of claim 14 wherein the label is colorimetric, radioactive, or fluorescent.
 19. The method of claim 14 wherein the label is a labeled quantum dot.
 20. The method of claim 14 further comprising sorting the solid phase supports from the labeled peptide library on the basis of label intensity.
 21. The method of claim 14 further comprising determining the sequence of the peptide attached to the solid phase support.
 22. The method of claim 14 further comprising determining the modification status of the peptide attached to the solid phase support.
 23. The method of claim 14 wherein the chemical modification comprises covalent modification of an amino acid.
 24. The method of claim 23 wherein the covalent modification comprises methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, or ADP ribosylation.
 25. The method of claim 14 wherein the protein is an enzyme.
 26. The method of claim 14 wherein the protein is an antibody.
 27. The method of claim 14 wherein the combinatorial library comprises an N-terminal peptide sequence from a histone.
 28. The method of claim 14 wherein the combinatorial library comprises an N-terminal peptide sequence from a histone, and wherein the protein is an antibody. 